1. Field of the Invention
The invention relates to the immunoassay of protein such as total and specific immunoglobulins in biological fluids and more specifically to methods and apparatus for such an immunoassay.
2. Brief Description of the Prior Art
Immunoglobulins are modified forms of the blood protein known as globulin and include antibodies and reagins. They are found in the body fluids of vertebrates following their sensitization by exposure to a protein or antigen foreign to the vertebrates evolutionary chemistry.
A wide variety of procedures are known for the immunoassay of immunoglobulins, i.e.; the determination of the quantity of immunoglobulins in a given biological fluid, using immuno-chemical technique. Immunochemistry is chemistry classically concerned with the physical interaction between "antigens" and "antibodies".
"Antigens" are high molecular weight compounds, usually protein or protein-polysaccharide complexes, which upon entry in the blood stream of a vertebrate stimulate the transformation of the small lymphocytes of the B-type into lymphoblasts. The lymphoblasts secrete antibodies specific to the antigen stimulator. The antibodies are proteins possessing reactive sites specifically complimentary to a reactive feature or site on the stimulating antigen. Antibodies generally have the property of rendering the antigen harmless to the host organism, by occupying the immunologically active sites on the antigen particles or molecules, and sometimes also by forcing precipitation or agglutination of the antigen, or by other protective mechanisms.
The words "precipitation" and "agglutination" differ in that precipitation refers to formation of a particulate or agglomerate solid from molecules initially in solution or, particularly in immunology, dissolved in blood serum. Agglutination refers to formation of an agglomerate from particulate substances initially suspended in a fluid. When suspended particles react with dissolved molecules to form an agglomerate, the term "agglutination" is usually applied. In addition, the word "agglomeration" is used as a general term encompassing both pairing of particles and clamping of a large multiplicity of particles.
In some applications it becomes difficult or meaningless to maintain the classical distinction between antigen and antibody, because in many regards the relation between antigen and antibody is reciprocal and each precipitates or agglutinates the other. The basis for the distinction resides in the history of the particular substances, and this can become irrelevant outside the original antibody-generating organism, for example in reagent applications. For this reason the antigen-antibody relationship may be advantageously described in this reciprocal way; an antibody is the "immunological homologue" of the antigen which produced it, and vice versa. An antibody and its corresponding antigen are thus homologues of each other. They may also be said to be homologous to each other.
In any event, the immunochemical antigen-antibody relationship forms the basis for immunoassay of either "homologue". Procedurally, the various known techniques of immunoassay for the immunoreactants (antigen, antibody), i.e.; radioimmunoassay, fluorescent immunoassay and enzyme immunoassay are substantially identical. Each technique comprises, in general, the separation of bound and labeled immunoreactant from unbound, labeled immunoreactant. This may be done, for example by immobilizing one of the immunoreactants, labeling one of the immunoreactants with a marker or tag to monitor its presence and reacting the immobilized immunoreactant with the free immunoreactant and measuring the degree of reaction through monitoring of the labeled immunoreactant. The difference between the various techniques resides in utilization of different reagents as markers or tags for visualization and measurement of the immunoreaction.
In the prior art immunoassay techniques, there are two principal methods for immobilizing one of the immunoreactants.
The first method of immobilization constitutes a physical adsorption or entrapment of the one immunoreactant on a water-insoluble solid such as charcoal, glass or a water-insoluble synthetic polymeric resin. The use of polymeric resins as the immobilizing substrate in the form of test tubes, microtiter plates, "dipsticks", or similar configurations is popular. The strong electrostatic charge of the resin substrate is advantageous and the procedure has proven satisfactory when measuring relatively high concentrations of unknown antigen or antibody, such as antibodies to specific infection where the antibody titers are high. However, the adsorption of immunoreagents onto plastic surfaces forms a significantly weaker bond than, for example covalent bonds. Washing steps in the procedure can often result in inaccurate or completely erroneous results by washing away the weakly bound immunoreagents. The loss of bound immunoreactant may adversely affect the immunoassay. Consider the following example:
OBJECTIVE: To determine if a patient's blood serum contains antibodies to rubella virus.
PROCEDURE:
1. Solid-phase+rubella protein (adsorbed). PA0 2. Add patient's serum--specific antibodies react with rubella protein. PA0 3. Wash extensively to remove extraneous immunoglobulin (IgG) in patient's serum. PA0 4. Add labeled (radioactive, fluorescent, or enzyme) anti-Human IgG (this reacts with IgG bound to rubella protein). PA0 5. Wash to remove unbound labeled anti-IgG. PA0 6. Monitor radiation remaining, fluorescence emitted, or add specific substrate to monitor enzyme activity. PA0 1. If the patient possesses rubella antibodies, Step 6 will produce a positive response. PA0 2. If the patient possesses no rubella antibodies, the washing will remove all of the labeled anti-IgG and Step 6 will give a negative response. PA0 1. If this occurred in Step 2, the patient's IgG could adsorb to the solid phase substrate. PA0 2. If this occurred to Step 3, labeled anti-IgG could adsorb to the solid phase substrate. PA0 3. If this occurred in Step 5, all proteins would be washed away. This would result in the observation of false negative reactions or false low titers.
INTERPRETATION:
CONSIDERATIONS: Suppose that the washing steps resulted in unexpected release of adsorbed rubella protein.
In both of these cases false positive reactions or false high titers would be observed.
The above-described assay generally presents no inaccuracy only when the immunochemical reactant being assayed is present in high concentrations. The losses are magnified several-fold however when the assay is to measure immunoreactants present in low concentrations.
In addition, the literature has pointed out the problems of nonspecific binding immunoreagents and unusually high response negative controls when using the adsorption procedure to immobilize immunoreactants for immunoassay procedures; see for example Sack, S. A., P. K. B. Neog:, M. D. Khorshed Alam. (1980), Immunobead Enzyme-Linked Immunosorbent Assay for Quantitating Immunoglobulin A in Human Secretions and Serum, Infection and Immunity, 12:281-283; Carlson, J., D. Gabel, E. Larsson, J. Ponten, B. Westermark (1979), Protein Coated Agarose Surface for Attachment of Cells, In Vitro, 15:844-850; and Boenisch, T. (1976). Improved Immunoassay for Trace Proteins In Proteins and Related Subjects, Protides of Biological Fluids, 24:743-749.
The second method of immobilizing an immunoreactant is by a covalent binding of the immunoreactant to a water-insoluble substrate. This is a very satisfactory method of immobilization, firmly holding the immunoreactant during washings, reaction etc. However, it will be appreciated that one is limited to the use of very specific, reactive substrates which will form a covalent bond with the immunoreactant. Representative of such substrates are diazotized polystyrene, p-aminobenzylcellulose (PAB-cellulose), isothiocyanate substituted graft copolymer of styrene and polytetrafluoroethylene and like materials. The disadvantages of such materials resides in the limited form available. For example, one of the most satisfactory of reactive solid-phase materials are the carbohydrate polymer beads and synthetic polymer beads such as agarose beads (sometimes activated with cyanogen bromide), carboxylated or aminated polyacrylamide beads and cellulose discs or particles activated with cyanogen bromide. However, these water-insoluble materials are not readily molded into forms other than beads, particles or discs and like forms. This is a disadvantage. The physical forms necessitated complicates the washing process during immunoassay in that the beads, discs or particles must be filtered, centrifuged, or allowed to settle between washes. Because of the nature of the cellulose discs and their greater absorbancy, they must be suspended in the wash solutions for an inordinately longer period of time. These are severe disadvantages when one desires rapid, efficient test procedures.
The present invention comprises a process for coating light-transparent, water-insoluble polymeric surfaces so that they remain optically clear yet become chemically reactive so that immunoreagents can be covalently bound to the surface. The light-transparent, water-insoluble polymeric resin so coated may be any of those moldable in configurations other than just beads, particles etc. (such as tubes, microtiter plates, petri dishes, and the like).
Also by the present invention, one may prepare water-insoluble surfaces normally inert to chemical reactions (glass, plastic, metal, and the like) for covalently bonding the primary immunoreagent in an immunoassay. This immunoreagent can be an antigen when the assay is for antibodies, or can be an antibody, lectin, or similar chemical when the assay is for an unknown protein or similar complex molecule.
We also describe herein a procedure for the use of the solid-phase technology described above in an immunoassay to monitor or measure total reaginic antibodies (immunoglobulin E of IgE) and/or allergen specific IgE utilizing enzyme immunoassay (EIA). Measurement of IgE is a specific case where the material to be assayed is present in extremely small concentrations (generally).
Reagins are complex organic compounds belonging to the class of immunoglobulins known as immunoglobulin E (generally referred to for convenience as "IgE"). More specifically reagins are a group of type IgE proteins found in the blood serum of vertebrates, following their sensitization by exposure to an allergen or allergens. The endogeneously produced reagin may be characterized in part by its antibody-like activity, i.e., its specific reactivity in binding at epitopic sites on the counterpart allergen which is the source of its own genesis. The reagin also generally has a propensity to attach to living cells throughout the body of the host vertebrate. When the counterpart allergen is reintroduced into the previously sensitized host vertebrate, an allergen-reagin reaction takes place usually with a consequential anaphylactoid type of immune reaction. The latter results primarily from a rupture of eosinophils having attached reagins--allergen complex. Rupture of the cells releases histamine, slow-reacting substance of anaphylaxis, eosinophil chemotaxic substance, lysosomal enzymes and other compounds which result in an allergic reaction in the host vertebrate. Allergic reactions include anaphylaxis, urticaria, hay-fever, asthma and like clinical manifestations.
To avoid allergen-reagin reactions in a sensitized vertebrate, one hopefully identifies reagins in the blood serum of the vertebrate and then precautions may be taken to limit exposure of the sensitized individual to allergens corresponding to the identified reagin or reagins or by desensitizing the individual to specific allergens.
In view of reagin-antibody activity, prior art in-vitro methods of identifying reagins in blood serum have been based, empirically, on the known and classic immunological relationship which exists between an antigen and its homologous antibody. However, such prior art methods have not been entirely satisfactory in regard to reagin identification for a number of reasons. First, allergens, which are in essence protein substances foreign to the chemistry of a given vertebrate, apparently stimulate the production of relatively small quantities of reagin in comparison for example to the production of antibody to disease antigens. The smaller production of reagin complicates its detection and identification in the complex mixture comprising blood serum.
Additionally, the majority of native allergens possess a plurality of allergenic determinants and when introduced into a vertebrate will provoke or elicit a mixed plurality of reagins instead of a single reagin. The mixture of reagins will differ from each other in their physicochemical and biological properties, complicating further identification of the reagin entity. Some of the minor compounds elicited in the mixture may be in such low concentrations that they are not detectable by conventional physicochemical techniques.
Secondly, since most IgE material isolated from host organisms has been found to be a heterogeneous mixture of structurally similar but diverse proteins, and a specific reagin may in fact be a mixture of different reagin molecules, any in-vitro detection method based on binding of the reagin with an allergen may depend for accuracy on a protocol which may not account for all of the diverse reagin molecules and not just a portion of the mixture.
In addition, it will be appreciated that since immune sera contains reagins which will bind to their corresponding allergens with varying degrees of avidity, strong positive allergen-reagin reactions may not always be obtained in reasonable times. Further, the physical nature of the reagin mixture might be expected to affect the strength of any interaction or binding of reagin which may occur.
It has also been recognized that IgE materials do not behave in the same way as, for example, IgM or IgG, the protective antibodies produced by an organism to counteract antigens related to diseases. In the latter process, the host organism may continue to produce "protective" types of antibody even after the disease state or entity has been eliminated, thereby obtaining immunity to re-infection. In contrast, in the case of allergy whose physical manifestation of the allergic response is the binding of the allergen with the reagin, no immunity is necessarily conferred. When the binding reaction occurs, cellular damage occurs wherein substances such as histamine are released to affect allergic target tissues. The binding reaction will occur during every subsequent re-introduction of allergen into the host organism.
Clearly, although there are apparent analogies between the classical immunological antigen-antibody relationship and the more specific allergen-reagin process, there are also subtle and marked differences. It is these differences which suggest that the prior art empirical use of antigen-antibody in-vitro identification procedures to identify allergens-reagins may have been misplaced and accounts for the inaccuracies which have been observed (lack of avidity, specificity) and the lack of sensitivity.
Because of the dissatisfactions with the prior art in-vitro methods of determining and identifying reagins in blood serums, the most widely employed methods of determining reagins present in the blood serum of allergen sensitized vertebrates (and thereby a differential diagnosis of atopic or anaphylactic allergy) are the in-vivo skin and provocation test methods. These in-vivo test methods are also lacking in complete satisfaction. They are time consuming, inconvenient to patients and not without serious risk. The potential for anaphylaxis upon exposure of the patient to allergens is a real hazard.
Like the prior art in-vitro methods for identifying reagins, the in-vivo methods are also inaccurate to a degree. The allergen-reagin reaction physical manifestations observed in skin-testing may be affected by subjective influences such as an allergic threshold in individual body resistance to allergic response. Emotional factors in the individual undergoing testing can also affect the allergic response.
In summary the prior art methods, both in-vitro and in-vivo, for identifying reagins in the blood sera of vertebrates have not been entirely reliable, accurate or safe for the variety of reasons described above. The method of the present invention is an improvement over the prior art methods.
There are a number of advantages associated with the method of our invention. A major advantage resides in the capability of performing allergen identification testing in the physician's office on a simple, economical and rapid basis. The use of the patient's blood serum in an in-vitro test method obviates the hazards associated with conventional skin-testing and provocation test procedures (risk of anaphylaxis). This is particularly advantageous where the very young, elderly and debilitated individual is the object of testing. Other advantages include more stable reagents with less associated hazards and which require less training in their use than those associated with, for example, the radioellergo-immunosorbent test (RAST) which employs radioactive labelled reagents. The reagents used in the method of the invention also have longer shelf-lives than radio-active labelled reagents and are safer to use.
The method of the invention also requires only relatively small blood serum samples for testing, providing the patient with considerably decreased discomfort and loss of time. Once the blood sample is obtained the patient need not wait for results. Automation of the procedure will enable the physician to increase the number of patients he can diagnose in a given time period.
Because the method of the invention measures total and specific IgE it enables the physician to monitor allergy therapy by monitoring serum IgE levels. This is a very sensitive monitor. In contra-distinction skin test results fluctuate rapidly over short periods of time depending on the physical state of the patient. The method of the invention is more consistent and repeatable than skin-testing because it comprises monitoring serum components which are less affected by the patient's physical condition.
Results of the immunoassay of the invention may be observed visually or quantitated with a simple spectrophotometer and highly sophisticated apparatus is not required.